During the activation and transferring process, E1 and E2 form a thioester-linkage with SUMOs. By using an in vitro assay, it is demonstrated that SENP1 is able to cleave the thioester-linkage between SUMO-1/SUMO-3 and E1/E2. This finding suggests that SUMO proteases regulate the sumoylation pathway, not only during maturation and deconjugation, but also in the E1 activation and E2 conjugation processes. / Recently, reactive oxygen species have been demonstrated to influence the equilibrium of sumoylation-desumoylation. Here, by in vitro assay, it is shown that H2O2 induces formation of inter-molecular disulfide linkage of human SUMO protease SENP1, via the active-site Cys 603 and a unique residue Cys 613. Such reversible modification confers higher enzyme activity recovery which is also observed in yeast Ulp1, but not in human SENP2, suggesting its protective role against irreversible sulfhydryl oxidation. The physiological relevance of the disulfide-linked dimer of SENP1 is also detected in cultured cells upon oxidative stress. The modifications are further verified by the crystal structures of Ulp1 with catalytic cysteine oxidized to sulfenic, sulfinic and sulfonic acids. The current findings suggest that, in addition to SUMO conjugating enzymes, SUMO proteases may act as redox sensors and effectors, which modulate the desumoylation pathway and allow immediate specific cellular responses to oxidative stress. / SUMO (small ubiquitin-related modifier) is a member of the ubiquitin-like protein family that is highly conserved in all eukaryotic organisms and regulates cellular function of a variety of target proteins. SUMO proteins are expressed in their precursor forms and precursor processing involves cleavage of the residues after the conserved 'GG' region by the hydrolytic activity of SUMO-specific protease. The exposed second glycine then forms a covalent bond with the epsilon-amino group of a substrate lysine residue at the psiKxE motif by a cascade of SUMO El, E2 and E3 ligases. As a reversible modification, SUMO proteases can cleave SUMOs from their substrates during de-conjugation process. / To date, four SUMO family members, SUMO-1, -2, -3 and -4 and six SUMO proteases, SENP1--3 and 5-7 (where SENP stands for sentrin-specific protease) have been identified in human. By characterizing the maturation reactions of SUMO-1, -2 and -3 catalyzed by SENP1, it is demonstrated that SENP1 contains the highest maturation efficiency for SUMO-1, followed by SUMO-2 and SUMO-3. By mutagenesis study, it is further identified that the two amino acids immediately after GG motif could influence the maturation efficiency of SENP1. By comparison with another investigation which showed the preference of the maturation reaction of SUMO-2 by SENP2, the results suggest that SUMO proteases with specific tissue distribution control the availability of different mature SUMOs in human. / To gain a deeper insight into the molecular basis of maturation and de-conjugation processes catalyzed by SENP1, it has been determined, at 2.8 A resolution, the X-ray structure of a complex between the catalytic domain of SENP1C(C603S) and matured SUMO-1. The structure shows that the substituted serine residue does not undergo any local structural rearrangements at the active site as observed in the previously solved SENP2/SUMO-1 complex structure. This finding suggests that SUMO proteases require a self-conformational change prior to the cleavage reaction, and further disclose the cleavage mechanism of the hydrolytic reactions catalyzed by SUMO proteases. Moreover, analysis of the interface of SENP1 and SUMO1 has identified four amino acids that are unique in SENP1 sequence and facilitate the interaction of SENP1 and SUMO-1. / Xu, Zheng. / "July 2007." / Advisers: Shannon Au Wing Ngor; Tzi-Bun Ng. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0125. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 181-194). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Les biofilms ont longtemps été décrits comme des organisations évolutives de microorganismes, attachés à une surface et englués dans une matrice contenant, entre autre, des polysaccharides. En partant de ce constat BioFilm Control a souhaité cribler des microorganismes pour la production d’exopolysaccharides, en utilisant le BioFilm Ring Test® (BRT). Le principe repose sur la coincubation de microorganismes avec des particules magnétiques en microplaque. Les particules sont plus ou moins attirées par un aimant en fonction du stade d’organisation du biofilm. En se formant, il piège, dans sa matrice visqueuse, les particules qui perdent leur mobilité. Celle-ci est révélée par une aimantation qui provoque l’apparition d’un spot (pas de biofilm) ou non (biofilm). Une analyse d’images quantifie ce processus et permet de le standardiser. La démarche a consisté dans un premier temps à vérifier le comportement de microorganismes modèles producteurs de polysaccharides (bactéries et microalgues) avec le BRT. L’étude a été étendue au criblage d’une banque de lactobacilles. Les résultats inattendus ont orienté l’étude vers l’analyse du rôle exact des polysaccharides et plus généralement de l’implication des macromolécules dans la structuration du biofilm. Pour cela, la dégradation séquentielle de chaque famille macromoléculaire a été réalisée via des enzymes dépolymérisantes sur les biofilms de Leuconostoc mesenteroides et Bacillus sp. Au regard des résultats obtenus, l’utilisation du BRT a été étendue à la caractérisation qualitative et quantitative d’enzymes de dégradation de polysaccharides. / Biofilms were described for a long time as evolutionary structures elaborated by microorganisms, fixed on a surface and maintained in a polysaccharidic matrix. From this assessment, BioFilm Control chose to screen microorganisms for their capacity to produce exopolysaccharides (EPS), using theBioFilm Ring Test® (BRT). The principle is the co-incubation of magnetic particles with microbial culture on microplates. The mobility of particles depends on the stage of biofilm formation. During this formation, particles are trapped in the matrix and loose their mobility. Revelation is induced by magnet which causes a spot in the absence of biofilm. The pictures analysis quantifies this phenomenon and standardizes different results. This approach was realised, at first step, by the test of EPS-producing bacteria or microalgae with the BRT. The study was extended to the screening of a lactobacilli collection. Unexpected results guided the research toward the understanding of the role of macromolecules in biofilm structuring. To study their implication, sequential enzymatic degradation has been achieved for each macromolecular family of Leuconostoc mesenteroïdes and Bacillus sp. biofilms. Using the results, BRT was then appreciated as a suitable method to detect and quantify polysaccharide degrading enzymes.

In this study, high-performance liquid chromatography (HPLC) was employed to detect and quantify pyrimidine salvage enzymes by monitoring the disappearance of substrates or formation of products.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The grape and wine ecosystem contains fungi, bacteria and yeasts whose interactions contribute to the final wine product. While the non-Saccharomyces yeasts are dominant in the early stage of alcoholic fermentation, the later stage is always dominated by Saccharomyces cerevisiae. Although their presence in wine fermentation is often short-lived, the non-Saccharomyces yeasts are known to produce an array of extracellular hydrolytic enzymes which facilitate the extraction and release of aroma compounds, but might also play a role in microbial interactions. The present study aimed to investigate the microbial diversity of grape juice and to evaluate the potential of non-Saccharomyces yeasts to produce hydrolytic enzymes and display anti-fungal properties. To capture the microbial diversity, culture-dependent (plating) and –independent (Automated Ribosomal Intergenic Spacer Analysis (ARISA)) techniques were used in parallel. The fungal and bacterial ARISA displayed a wider range of operational taxonomic units (OTUs) in comparison to cultivation-based technique, demonstrating that ARISA is a powerful culture-independent technique applicable to ecological studies in wine. Some of the uncommon yeast isolates derived from our cultivation-based study were subjected to an enzymatic screening process. Hydrolases, such as chitinases, β-1,4-cellulases, β-1,3-1,6-glucanases, β-glucosidases, pectinases and acid proteases were specifically sought. Most of the yeast isolates exhibited chitinase, β-1,4-cellulase as well as β-1,3-1,6-glucanase activities. Only Metschnikowia chrysoperlae exhibited β-glucosidase activity. We also retrieved the partial chitinase gene sequences from M. chrysoperlae, Pichia burtonii, Hyphopichia pseudoburtonii that exhibited chitinase activity. Among the isolates, Pseudozyma fusiformata exhibited a strong antagonistic activity against the wine spoilage yeasts B. bruxellensis AWRI 1499 and B. anomalus IWBT Y105. Furthermore, we showed that the killer phenotype of P. fusiformata cannot be attributed to a viral encoded dsRNA. Finally, two metagenomic approaches were employed in an attempt to explore the indigenous microbiome in a more holistic manner, where we adopted whole metagenome Roche GS-FLX 454-pyrosequencing and construction of a fosmid library. The whole metagenome sequencing revealed a wide range of hydrolytic enzymes that showed homology to enzymes from different fungal and non-Saccharomyces yeast species. Moreover, the metagenomic library screening resulted in the retrieval of 22 chitinase and 11 β-glucosidase positive fosmid clones originating from yeasts. Two clones of interest, BgluFos-G10 and ChiFos-C21, were subjected to next generation sequencing. BgluFos-G10 revealed 2 ORFs exhibiting homology to glycosyl hydrolase family 16 proteins whereas no ORFs encoding chitinase enzymes could be identified in the ChiFos-C21 clone. However, all the potential ORFs identified exhibited homology to a gene cluster from Clavispora lusitaniae ATCC 42720, suggesting that the cloned DNA fragments belonged to a yeast species closely related to C. lusitaniae or members of the family Metschnikowiaceae. Overall, our study identified a variety of novel hydrolytic enzymes. However, retrieving the full gene sequences of these identified enzymes would be the immediate follow-up of our study. Moreover, the hydrolytic and antifungal activities exhibited by the yeast isolate could be of major interest in evaluating their potential as biocontrol agents against grapevine fungal pathogens and subsequently the wine spoilage yeasts. It would be interesting to evaluate as well the potential impact of these enzymes under wine making condition and could be our next step of investigation. / AFRIKAANSE OPSOMMING: Die druif en wyn ekosisteme bevat swamme, bakterië en giste en die interaksies van hierdie organismes dra by tot die finale wyn produk. Die nie-Saccharomyces giste is dominant in die vroeë stadium van die alkoholiese fermentasie, maar die latere fase word altyd gedomineer deur Saccharomyces cerevisiae. Alhoewel hulle teenwoordigheid in wyngistings gewoonlik kortstondig is, is die nie-Saccharomyces giste bekend vir die produksie van ‘n verskeidenheid ekstrasellulêre hidrolitiese ensieme wat die ekstraksie en vrylating van aroma komponente fasiliteer, en ook moontlik ‘n rol kan speel in mikrobiese interaksie. Hierdie studie beoog om die mikrobiese diversiteit van druiwesap te bestudeer en die potensiaal van nie-Saccharomyces giste te evalueer ten opsigte van die produksie van hidrolitiese ensieme, asook die demonstrasie van anti-swam eienskappe. Kweking-afhanklike (uitplating), asook –onafhanklike (Automatiese Ribosomale Intergeniese Spasieerder Analise (ARISA)) tegnieke is in parallel gebruik om die mikrobiese diversiteit te bepaal. Die swam en bakteriële ARISA het ‘n groter verskeidenheid van operasionele taksinomiese eenhede (OTUe) vertoon in vergelyking met die kweking-gebasseerde tegniek en dit demonstreer dat ARISA ‘n kragtige kweking-onafhanklike tegniek is, wat toepasbaar is in ekologiese studies van wyn . Sommige van die skaarser gisisolate, uit ons kweking -gebasseerde studie was vir ensiemaktiwiteite geskandeer. Daar is spesifiek gesoek vir hidrolases soos chitinases,β-1,4-sellulases, β-1,3-1,6-glukunases, β-glukosidases, pektinases en suur proteases. Die meeste gisisolate het chitinase,β-1,4-sellulase asook β-1,3-1,6-glukunase aktiwiteit vertoon. Slegs Metschinikowia chrysoperlae het β-glukosidase aktiwiteit vertoon. Ons het verder die gedeeltelike chitinase geensekwensies van M. chrysoperlae, Pichia burtonii en Hyphopichia pseudoburtonii wat chitinase aktiwiteit vertoon het, bepaal. Een isolaat, Pseudozyma fusiformata, het ‘n sterk antagonistiese aktiwiteit teenoor die wyn bederfgiste, Bretanomyces bruxellensis AWRI 1499 en B. anomalus IWBT Y105 vertoon. Verder het ons gewys dat die killer fenotipe van P. fusiformata nie gekoppel kan word aan’n viraal gekodeerde dsRNA nie. Ten laaste is twee metagenomiese benaderings, naamlik die volledige metagenoom Roche GS-FLX 454-pirovolgordebepaling en konstruksie van ‘n fosmied biblioteek, gebruik om die inheemse mikrobioom op ‘n meer holistiese wyse te bestudeer. Die volgordebepaling van die volledige metagenoom het ‘n wye verskeidenheid hidrolitiese ensieme aan die lig gebring wat homologie met ensieme van verskillende swamme en nie-Saccharomyces gisspesies getoon het. Verder het die skandering van die metagenomiese biblioteek die isolasie van fosmiedklone van gisoorsprong wat positief is vir chitinase aktiwiteit (22 klone) en β-glukosidase aktiwiteit (11 klone) tot gevolg gehad. Twee van hierdie klone, BgluFos-G10 en ChiFos-C21, is met volgende generasie volgordebepaling ontleed. BgluFos-G10 het twee oopleesrame (OLRe) wat homologie met glikosiel hidrolase familie 16 proteïene het, vertoon maar geen OLRe wat chitinase ensieme enkodeer kon in die ChiFos-C21 kloon geïdentifiseer word nie. Al die potensiële OLRe wat geïdentifiseer is, het homologie aan ‘n genepoel van Clavispora lusitaniae ATCC 42720 vertoon, wat daarop dui dat die gekloneerde DNS fragmente aan ‘n gisspesie behoort wat naverwant aan C. lusitaniae of lede van die Metschinikowiaceae familie is. In geheel gesien het ons studie ‘n verskeidenheid van nuwe hidrolitiese ensieme geïdentifiseer. Die bepaling van die volledige geenvolgordes van hierdie geïdentifiseerde ensieme sal die onmiddelike opvolg aksie van hierdie studie wees. Verder is die hidrolitiese en anti-swam aktiwiteite wat deur die gisisolate gedemonstreer is, van hoof belang, asook die evaluering van hulle potensiaal as biokontrole agente teen wingerd swampatogene en wyn bederfgiste. Dit sal ook interessant wees om die potensiële impak van hierdie ensieme onder wynmaakkondisies te bepaal, en dit kan dus ons volgende ondersoek stap wees.

The clcD gene encoding dienelactone hydrolase (DLH) is part of the clc gene cluster for the utilization of the B-ketoadipate pathway intermediate chlorocatechol. The roles that individual amino acids residues play in catalysis and binding of the enzyme were investigated. Using PCR a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHi to ZscoRI subclone in the plasmid pUC19.

A proteína recombinante inibidora de serinoprotease denominada de Amblyomin-X foi obtida a partir de uma biblioteca de cDNA das glândulas salivares do carrapato Amblyomma cajannense, construída e utilizada para identificar um gene que codifica um inibidor de serinoprotease do tipo Kunitz. O Amblyomin-X inibe a formação da massa tumoral in vivo, no entanto o mecanismo envolvido neste efeito não está totalmente esclarecido. Visto que um dos mecanismos anti-carcinogênicos dos inibidores de serinoproteases é a inibição do processo de angiogênese, este trabalho foi delineado para avaliar as ações do Amblyomin-X sobre a angiogênese in vivo e sobre funções da célula endotelial envolvidas neste processo. A angiogênese in vivo foi estudada em modelo de câmara dorsal por microscopia intravital. Quarenta e oito horas após a implantação da câmara dorsal, os animais receberam tratamento tópico de salina ou de Amblyomin-X por 8 dias, com intervalos de 48 horas a cada dose (10, 100 ou 1000ng/mL). Os efeitos foram avaliados em condições basais e na vigência do crescimento tumoral (injeção de 1x105 células B16-F10 de melanoma murino no tecido subcutâneo). Adicionalmente, os efeitos do Amblyomin-X sobre a permeabilidade vascular foram avaliados pela mensuração espectrofotométrica da quantidade de corante extravasado no tecido dos animais após injeção intradérmica do fator de crescimento do endotélio vascular (VEGF) ou do Amblyomin-X. Uma série de estudos in vitro foram realizados em células endoteliais de linhagem de microcirculação (t-End) para avaliar os efeitos do Amblyomin-X (10, 100 e 1000ng/mL) sobre: 1) a migração destas células, usando modelos bidimensional (2D) de cicatrização in vitro e tridimensional (3D) em câmara de Boyden modificada, na ausência e frente ao fator de crescimento do endotélio vascular (VEGF; 100 ng/mL); 2) sobre a aderência em Matrigel® e 3) sobre a secreção de prostaglandina E2 (PGE2) e a produção de óxido nítrico (NO) por ensaio imunoenzimático e reação de Griess, respectivamente. Ademais, foram avaliados os efeitos do Amblyomin-X sobre a viabilidade das células B16-F10 (1x105) por citometria de fluxo. Os resultados obtidos mostram que a aplicação tópica de Amblyomin-X reduziu o número de vasos no tecido subcutâneo dorsal (10ng/mL = 21,7%; 100ng/mL= 35,7%; 1000ng/mL= 36,8% vs 1° dia de tratamento). O mesmo efeito foi observado na presença de células B16-F10 (1000ng/mL= 44,3% vs 1° dia de tratamento), além de uma redução no desenvolvimento da massa tumoral (1000ng/mL= 88% vs controle). O tratamento com Amblyomin-X reduziu a migração basal das células t-End no modelo 2D (10ng/mL=16,4%; 1OOng/mL=23, 1%; 1000ng/mL=26,8% vs controle) e 3D (10ng/mL=39,2%; 100ng/mL=49,4%; 1000ng/mL=50,4% vs controle); inibiu a adesão destas células endoteliais em Matrigel® (100ng/mL=46,4%; 1000ng/mL=48,4% vs controle); não alterou produção os mediadores químicos NO e PGE2 pelas células endoteliais; não modificou a permeabilidade vascular e não alterou a viabilidade das células de melanoma murino B16-F10. Em conjunto, os dados obtidos mostram que o Amblyomin-X inibe a formação de novos vasos em condições basais e na vigência de crescimento tumoral in vivo que este efeito pode estar relacionado à redução do desenvolvimento tumoral, uma vez que a concentração de Amblyomin-X que inibe a angiogênese não causou citotoxicidade às células tumorais in vitro. Além disso, os mecanismos envolvidos no processo de angiogênese podem ser decorrentes, pelo menos em parte, de prejuízos na migração e adesão das células endoteliais. / The recombinant serine protease inhibitor protein called Amblyomin-X was obtained from a cDNA library of the Amblyomma cajennense salivary glands constructed and used to identify a gene encoding a kunitz type serine protease inhibitor. Amblyomin-X presents inhibitory effect on tumoral mass formation in vivo. Nevertheless, the mechanisms involved in the effects have not been clarified. Considering that interference on angiogenesis process is one of the mechanisms responsible for the antitumor activity displayed by serine protease inhibitors, this project was undertaken to study the Amblyomin-X actions on this process and on related endothelial cell functions. In vivo angiogenesis was studied using dorsal chamber model associated to intravital microscopy. Forty eight hours after dorsal chamber implantation, the animals were topically treated with saline or Amblyomin-X during 8 days, with intervals at each 48hs (10, 100 ou 1000ng/mL). The effects were evaluated at basal conditions or during tumoral development (1x105 B16-F10 murine melanoma cells injected into subcutaneous tissue). In addition, the effects of Amblyomin-X on vascular permeability were evaluated by measuring the dye leakage into dorsal intradermic tissue after local injection of vascular endothelial growth factor (VEGF) or Amblyomin-X, or both. In vitro assays were also performed using endothelial cells from microcirculation (t-End) and the effects of Amblyomin-X (10, 100 e 1000ng/mL) were studied on: 1) cell migration, using bidimensional (2D) and tridimensional (3D) models in modified Boyden chamber using chemotatic factor (VEGF100 ng/mL); 2) Matrigel® adherence and, 3) prostaglandin E2 (PGE2) and nitric oxide (NO) secretion by enzymatic assay and Griess reaction, respectively. In addition, the Amblyomin-X toxicity was evaluated on the B16-F10 cells (1x105), using flow citometry. Results obtained show that topic application of Amblyomin-X reduced the number of vessels in the subcutaneous dorsal tissue (10ng/mL = 21,7%; 100ng/mL= 35,7%; 1000ng/mL= 36,8% vs 1st day of treatment). The same effect was observed in the presence of B16-F10 cells (1000ng/mL= 44,3% vs 1st day of treatment), simultanesouly to a significant reduction on tumoral mass development (1000ng/mL= 88% vs control). Amblyomin-X treatment impaired basal migration of tEnd in the 2D (10ng/mL=16,4%; 100ng/mL=23, 1%; 1000ng/mL=26,8% vs control) and 3D model (10ng/mL=39,2%; 100ng/mL=49,4%; 1000ng/mL=50,4% vs controle); inhibited the adhesion of t-End in Matrigel® (100ng/mL=46,4%; 1000ng/mL=48,4% vs control); did not alter the production of chemical mediators (PGE2 and NO); did not modify the vascular permeability and did not affect the B16-F10 cells viability. Taken together, data here obtained show that Amblyomin-X inhibited the new vessels formation under basal conditions, and during tumoral development. The effect could be related to the reduction of tumoral progress also detected in vivo, asthe schedule of treatment employed did not induce cancer cell toxicity. The mechanisms involved in the reduced angiogenesis may be related, at least in part, to the impaired endothelial cell migration and adhesion.

Fung Pik Ha. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 116-121). / Acknowledgments --- p.i / Table of Contents --- p.ii / List of Figures --- p.v / List of Tables --- p.x / Abstract --- p.xi / Chapter Chapter I --- Introduction --- p.1 / Chapter Chapter II --- Literature Reviews / Chapter II.l --- Glycosidase --- p.3 / Chapter II.2 --- Biosynthesis of N-linked Glycoprotein --- p.4 / Chapter II.3 --- Mechanism of Enzyme Catalysed Reaction --- p.8 / Chapter II.4 --- Types of Glycosidase Inhibitors --- p.12 / Chapter II.5 --- Cyclophellitol and Aminocyclitols / Chapter II.5.1 --- General background on cyclophellitol --- p.17 / Chapter II.5.2 --- Mode of inhibition of cyclophellitol --- p.20 / Chapter II.5.3 --- General background on aminocyclitols --- p.24 / Chapter Chapter III --- Characterization of Synthetic Glycosidase Inhibitors / Chapter III.1 --- Covalent-based Inactivator (Cyclophellitol and its Analogues) / Chapter III.1.1 --- Introduction --- p.28 / Chapter III.1.2 --- Materials --- p.32 / Chapter III.1.3 --- Methods / Chapter III.1.3.1 --- Inhibitory assay of commercially available glycosidases --- p.33 / Chapter III.1.3.2 --- Partial purification of β-D-mannosidase from A. oryzae --- p.34 / Chapter III.1.3.3 --- Protein assay in purification of β-D-mannosidase --- p.38 / Chapter III.1.3.4 --- Inhibitory assay for partially purified β-D- mannosidase (A . oryzae) --- p.38 / Chapter III.1.3.5 --- Influence of dialysis on glycosidase inhibition --- p.39 / Chapter III.1.3.6 --- Inactivation experiment on glycosidases --- p.39 / Chapter III.1.4 --- Results / Chapter III.1.4.1 --- Inhibitory activities of cyclophellitol and its analogues against glycosidases --- p.41 / Chapter III.1.4.2 --- Effect of dialysis on glycosidase inhibition --- p.44 / Chapter III.1.4.3 --- The kinetic studies of glycosidase inactivation --- p.47 / Chapter III. 1.5 --- Discussion --- p.50 / Chapter III.1.6 --- Further studies --- p.55 / Chapter III.2 --- Reversible Competitive Inhibitors (Aminocyclitols) / Chapter III.2.1 --- Introduction --- p.56 / Chapter III.2.2 --- Materials --- p.58 / Chapter III.2.3 --- Methods / Chapter III.2.3.1 --- Assay of glucoside hydrolase inhibition activity --- p.60 / Chapter III.2.3.2 --- Glucose oxidase method for determination of released D-glucose --- p.60 / Chapter III.2.3.3 --- Inhibitory assay of aminocyclitols on other glycosidases --- p.61 / Chapter III.2.3.4 --- Influence of dialysis on the glycosidase inhibition --- p.62 / Chapter III.2.3.5 --- Lineweaver-Burk plot --- p.63 / Chapter III.2.4 --- Results / Chapter III.2.4.1 --- Inhibitory activities of valiolamine and related aminocyclitols against six glycosidases --- p.64 / Chapter III.2.4.2 --- Characterization the aminocyclitols as reversible competitive inhibitors --- p.69 / Chapter III.2.5 --- Discussion --- p.80 / Chapter Chapter IV --- Isolation of the Naturally Occurring Glycosidase Inhibitor from Mushrooms / Chapter IV.1 --- Introduction --- p.83 / Chapter IV.2 --- Materials --- p.84 / Chapter IV.3 --- Methods / Chapter IV.3.1 --- Preparation of Ganoderma lucidum --- p.86 / Chapter IV.3.2 --- Preparation of V. volvacea --- p.86 / Chapter IV.3.3 --- Inhibitory assay of aqueous extract of mushrooms on glycosidases --- p.87 / Chapter IV.3.4 --- Anthrone method for determination of reducing sugars --- p.87 / Chapter IV.3.5 --- Flash liquid chromatography for purification of putative inhibitors in G. lucidum --- p.88 / Chapter IV.4 --- Results / Chapter IV.4.1 --- Prescreening of Inhibitory effects of Various Fungal Extracts --- p.90 / Chapter IV.4.2 --- Inhibitory Effects of Partially Purified G. lucidum Extract on Glycosidase --- p.92 / Chapter IV.4.3 --- Effect of Endogenous Substrates on Glycosidase Activities --- p.93 / Chapter IV.4.4 --- Results of Liquid Column Chromatography --- p.93 / Chapter IV.4.5 --- Structure Determination and Characterization of purified compounds --- p.95 / Chapter IV.4.6 --- Inhibitory Activities of Compounds A and B against Brewers yeast a- glucosidase --- p.96 / Chapter IV.5 --- Discussion --- p.98 / Chapter Chapter V --- Conclusions --- p.113 / References --- p.116

Recent developments in NMR hardware have extended the reach of natural products chemists to structure elucidation of compounds isolated on a nanomolar scale. In parallel with advances in hardware new NMR techniques for structure elucidation have evolved, e.g. quantitative NOEs, residual dipolar couplings (RDCs)and diffusion-ordered spectroscopy (DOSY). The application of recent NMR techniques was utilized in the screening and structural assignment of natural products isolated from a 2004 collection of South African tunicates. Assignment of the planar structures of mandelalides A-D from a new Lissoclinum sp. was feasible only after data acquisition on a 700 MHz magnet equipped with 5 mm ����C cryogenic probe. While relative configuration of the polyketide mandelalides was established after extensive J-coupling and NOE analysis, quantitative ROESY and RDC measurements were also explored before the absolute configuration was accomplished by chemical degradation and comparison with standards. The application of DOSY, a greatly under-appreciated technique in natural products research, led to the identification of new rubrolide analogues, compounds with moderate antibacterial properties from the new species Synoicum globosum. Finally, relative and absolute configuration of new and known unstable amino alcohols from two Pseudodistoma species was based on J-coupling analysis and application of the Mosher method. / Graduation date: 2013

The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.

The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.